Fig. 7: Loss of exon 6 containing Runx1 isoforms impacts HSC emergence.

a, Venn diagram showing the intersect between isoform-based entropy and dIF level differences between HE^AGM and one or both extra-embryonic HE populations as well genes found to upregulated in the HE^AGM (as shown in Fig. 6a) (left). Gene Ontology analyses across Wikipathways 2024 Mouse, KEGG 2021 human and MSigDB Hallmark 2020 databases (right). The input gene lists consisted of the 1,579 genes that showed differential isoform expression (entropy and/or dIF) between HE^AGM and one or both YS HE. Gene lists can be interrogated in Supplementary Table 4. Adjusted P values were calculated using Fisher’s exact test with Benjamini–Hochberg correction b, Venn diagram depicting the intersects between genes upregulated in the HE^AGM (as depicted in Fig. 6a), the shared HE profile (as depicted in Fig. 5a) and the 210 gene list of transcription and chromatin factors with distinct HE^AGM isoform expression profiles (left). Cell identity analysis performed on all transcription and chromatin factors present in the list of genes with potential HE^HSC-selective isoform expression (middle). Adjusted P values were calculated using Fisher’s exact test with Benjamini–Hochberg correction. Venn diagram intersect for the stemness genes identified in the cell identity analyses (right). The diagram shows if the genes were selected based on changes in Entropy or dIF. c, Emerging hematopoietic cells (CD31^posKIT^posCD45^pos) in E11.5 WT and CRISPR-Cas9 Runx1 Δ exon 6 embryos identified by flow cytometry (Extended Data Fig. 1d). Percentage of emerging hematopoietic cells in E11.5 AGM regions (top). Each point represents a single AGM. Percentage of emerging hematopoietic cells in E11.5 YSs (bottom). Each point represents a single YS. WT n = 11, Δ exon 6 n = 7. Bars represent the average percentage of emerging hematopoietic cells ± s.e.m. Statistical test used was an unpaired two-tailed t-test. d, Phenotypic LT HSCs in E11.5 AGM and E16 FL identified by flow cytometry (Extended Data Fig. 1d). Left, percentage of LT HSCs in E11.5 AGMs. Each point represents a single AGM (WT n = 10, Δ exon 6 n = 7). Right, percentage of LT HSCs in E16 FLs. Each point represents a single FL (WT n = 11, Δ exon 6 n = 11). Bars represent the average percentage of LT HSC ± s.e.m. Statistical test used was an unpaired two-tailed t-test. e, Schematic of E14 FL LT HSCs transplantation experiments (top). A total of 150 phenotypic FL LT HSCs (CD45.2) were transplanted into sublethally irradiated NSGS mice (CD45.1). Donor contribution was followed for 12 weeks. Peripheral blood was analyzed by flow cytometry 4, 8 and 11 weeks post-transplant. At 12 weeks post-transplant the bone marrow (BM) was collected and analyzed by flow cytometry. Contribution of donor cells (CD45.2) to the peripheral blood of the transplanted mice at week 4 (WT n = 4, Δ exon 6 n = 5), 8 (WT n = 3, Δ exon 6 n = 4) and week 11 (WT n = 3, Δ exon 6 n = 3) (bottom). Bars represent the average percentage of donor derived blood cells  ± s.e.m. Unpaired two-tailed t-test. f, Bar graphs presenting the percentage of donor derived cells in the BM of recipient mice 12 weeks post-transplant. Donor cell contribution to the total BM (left). Donor cell contribution to the lineage negative (TER-119^negCD3^negB220^negGR1^negMAC1^neg) BM population (middle). Donor cell contribution to the BM LSK (TER-119^negCD3^negB220^negGR1^negMAC1^negSCA1^posKITt^pos) population (right). n = 3. Bars represent the average percentage of donor derived blood cells ± s.e.m. Unpaired two-tailed t-test. g, Myeloid (GR1^pos and/or MAC1^pos), B cell (CD19^pos) and T cell (CD3^pos) lineage output of donor cells in recipient BM 12 weeks post-transplant. n = 3. Bars represent the average percentage of lineage contribution of donor derived blood cells  ± s.e.m. Unpaired two-tailed t-test.

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